Cocrystal Applications in Drug Delivery Printed Edition of the Special Issue Published in Pharmaceutics www.mdpi.com/journal/pharmaceutics Andrea Erxleben Edited by Cocrystal Applications in Drug Delivery Cocrystal Applications in Drug Delivery Editor Andrea Erxleben MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin Editor Andrea Erxleben National University of Ireland Galway Ireland Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal Pharmaceutics (ISSN 1999-4923) (available at: https://www.mdpi.com/journal/pharmaceutics/ special issues/cocrystal applications). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year , Volume Number , Page Range. 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Contents About the Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Andrea Erxleben Cocrystal Applications in Drug Delivery Reprinted from: Pharmaceutics 2020 , 12 , 834, doi:10.3390/pharmaceutics12090834 . . . . . . . . . 1 Ricardo Machado Cruz, Tereza Boleslavsk ́ a, Josef Ber ́ anek, Eszter Tieger, Brendan Twamley, Maria Jose Santos-Martinez, Ondˇ rej Dammer and Lidia Tajber Identification and Pharmaceutical Characterization of a New Itraconazole Terephthalic Acid Cocrystal Reprinted from: Pharmaceutics 2020 , 12 , 741, doi:10.3390/pharmaceutics12080741 . . . . . . . . . 5 Ilma Nugrahani, Rizka A. Kumalasari, Winni N. Auli, Ayano Horikawa and Hidehiro Uekusa Salt Cocrystal of Diclofenac Sodium-L-Proline: Structural, Pseudopolymorphism, and Pharmaceutics Performance Study Reprinted from: Pharmaceutics 2020 , 12 , 690, doi:10.3390/pharmaceutics12070690 . . . . . . . . . 23 Aneta Wr ́ oblewska, Justyna ́ Sniechowska, Sławomir Ka ́ zmierski, Ewelina Wielgus, Grzegorz D. Bujacz, Grzegorz Mlosto ́ n, Arkadiusz Chworos, Justyna Suwara and Marek J. Potrzebowski Application of 1-Hydroxy-4,5-Dimethyl-Imidazole 3-Oxide as Coformer in Formation of Pharmaceutical Cocrystals Reprinted from: Pharmaceutics 2020 , 12 , 359, doi:10.3390/pharmaceutics12040359 . . . . . . . . . 51 Xavier Buol, Koen Robeyns, Camila Caro Garrido, Nikolay Tumanov, Laurent Collard, Johan Wouters and Tom Leyssens Improving Nefiracetam Dissolution and Solubility Behavior Using a Cocrystallization Approach Reprinted from: Pharmaceutics 2020 , 12 , 653, doi:10.3390/pharmaceutics12070653 . . . . . . . . . 73 Reynaldo Salas-Z ́ u ̃ niga, Christian Rodr ́ ıguez-Ruiz, Herbert H ̈ opfl, Hugo Morales-Rojas, Obdulia S ́ anchez-Guadarrama, Patricia Rodr ́ ıguez-Cuamatzi and Dea Herrera-Ruiz Dissolution Advantage of Nitazoxanide Cocrystals in the Presence of Cellulosic Polymers Reprinted from: Pharmaceutics 2020 , 12 , 23, doi:10.3390/pharmaceutics12010023 . . . . . . . . . . 89 Dnyaneshwar P. Kale, Vibha Puri, Amit Kumar, Navin Kumar and Arvind K. Bansal The Role of Cocrystallization-Mediated Altered Crystallographic Properties on the Tabletability of Rivaroxaban and Malonic Acid Reprinted from: Pharmaceutics 2020 , 12 , 546, doi:10.3390/pharmaceutics12060546 . . . . . . . . . 107 Bwalya A. Witika, Vincent J. Smith and Roderick B. Walker A Comparative Study of the Effect of Different Stabilizers on the Critical Quality Attributes of Self-Assembling Nano Co-Crystals Reprinted from: Pharmaceutics 2020 , 12 , 182, doi:10.3390/pharmaceutics12020182 . . . . . . . . . 129 Bwalya A. Witika, Vincent J. Smith and Roderick B. Walker Quality by Design Optimization of Cold Sonochemical Synthesis of Zidovudine-Lamivudine Nanosuspensions Reprinted from: Pharmaceutics 2020 , 12 , 367, doi:10.3390/pharmaceutics12090834 . . . . . . . . . 145 v About the Editor Andrea Erxleben obtained her PhD in Chemistry from the University of Dortmund (now TU Dortmund) in 1995. Following postdoctoral work with Professor Jik Chin at McGill University, Montreal, Canada, she returned to Dortmund where she completed her “habilitation” and obtained the “venia legendi” for Inorganic Chemistry in 2002. She moved to the National University of Ireland Galway where she is now a Senior Lecturer. She held guest professorships at the University of Vienna and is a member of the Editorial Board of Pharmaceutics and the Editorial Advisory Board of Crystal Growth and Design Her research interests are in the fields of Medicinal Inorganic Chemistry and Pharmaceutical Solid-State Chemistry. vii pharmaceutics Editorial Cocrystal Applications in Drug Delivery Andrea Erxleben 1,2 1 School of Chemistry, National University of Ireland, H91TK33 Galway, Ireland; andrea.erxleben@nuigalway.ie 2 Synthesis and Solid State Pharmaceutical Centre (SSPC), V94T9PX Limerick, Ireland Received: 21 August 2020; Accepted: 29 August 2020; Published: 1 September 2020 Over the past two decades, considerable research e ff orts in academia and industry have gone into pharmaceutical cocrystals [ 1 , 2 ]. As a result, a large number of studies on both fundamental aspects and applications of cocrystallisation have been published, and a few cocrystals are now on the market or in clinical trial phases, e.g., sacubitril-disodium valsartan-water (Entresto TM ), escitalopram oxalate-oxalic acid (Lexapro ® ), ertuglifozin-L-pyroglutamic acid and tramadol-celecoxib. The FDA defines pharmaceutical cocrystals as “crystalline materials composed of two or more di ff erent molecules, typically active pharmaceutical ingredient (API) and cocrystal formers (‘coformers’), in the same crystal lattice” [ 3 ]. Cocrystallisation is an attractive strategy to modify and improve the physicochemical properties of an API without making covalent changes to the drug molecule itself. Very often cocrystals are designed to tackle the poor dissolution behaviour and low bioavailability of Biopharmaceutics Classification System (BCS) class II and IV drugs that make up 70% of the drug candidates in the development pipeline [ 4 ]. However, chemical stability, hygroscopicity, mechanical, and flow properties have also been improved through cocrystal formation [ 1 , 2 ]. Furthermore, cocrystallisation can be used as a purification and enantiomeric separation method [5]. This Special Issue includes eight original research articles that highlight the relevance and versatility of pharmaceutical cocrystals in drug delivery. Machado Cruz et al. report a new cocrystal of the poorly water-soluble antifungal agent itraconazole [ 6 ]. They carried out a comprehensive study of the solid-state properties and the formation of the itraconazole-terephthalic acid cocrystal. The cocrystal is stable in aqueous solution and comparison with previously described itraconazole cocrystals showed a correlation of the intrinsic and powder dissolution rates with the solubility of the coformer. The dissolution behaviour of physical mixtures of the cocrystal and common excipients was also analysed. Nugrahani et al. prepared the mono- and tetrahydrate of the salt cocrystal diclofenac sodium- l -proline [ 7 ]. The hydrates were characterised by single crystal X-ray analysis and were shown to have higher solubilities and dissolution rates than the sodium salt of diclofenac acid and the anhydrous diclofenac acid- l -proline cocrystal. The release of water on drying led to the dissociation of the salt cocrystal into a physical mixture of diclofenac acid and L-proline. Interestingly, this process was reversible. When the dried sample was kept at 72% relative humidity and 25 ◦ C, diclofenac sodium-L-proline tetrahydrate was restored. A paper by Buol et al. describes the first cocrystals of the nootropic drug nefiracetam [ 8 ]. A large cocrystal screen with 133 di ff erent coformers using liquid-assisted grinding led to the identification of 13 new cocrystals that were characterised by single-crystal X-ray di ff raction. The study illustrates how solid-state properties—in this case, the melting point—can be varied over a wide range by changing the coformer. Three cocrystals with biocompatible coformers were subjected to a more comprehensive screen including solvent evaporation, slurrying and cooling crystallisation. The discovery of additional solid-state forms demonstrates the importance of a thorough screening for multiple cocrystal forms. The solubilities and dissolution properties of the new cocrystals were investigated. Kale et al. studied the e ff ect of cocrystallisation on the tabletability of rivaroxaban and found an improved tabletability for rivaoxaban-malonic acid [ 9 ]. The tabletability order malonic acid < Pharmaceutics 2020 , 12 , 834; doi:10.3390 / pharmaceutics12090834 www.mdpi.com / journal / pharmaceutics 1 Pharmaceutics 2020 , 12 , 834 rivaboxaban < rivaboxaban-malonic acid could be rationalised with the crystal packing, specifically the absence or presence of slip planes, slip plan topology, the degree of intermolecular interactions and d-spacing. Rivaroxaban contains slip planes with a flat-layered topology and with a zigzag layer topology. The crystal structure-mechanical property relationships found in this study shine light on the way crystals that contain more than one slip-plane system deform. Wroblewska et al. introduced the new coformer 1-hydroxy-4,5-dimethyl-imidazole 3 oxide [ 10 ]. They used high resolution-solid-state NMR to investigate the cocrystal formation with barbituric and thiobarbituric acid during ball-milling. The structures of the new coformer and cocrystals were studied by 13 C CP / MAS, 15 N CP / MAS and 1 H Very Fast MAS NMR in combination with single-crystal X-ray analysis. The e ff ect of the polymorphic and tautomeric form of barbituric / thiobarbituric acid on the cocrystallisation was evaluated. The cocrystals showed no cytotoxicity in HeLa and 293T cells at concentrations of up to 100 μ M indicating good biocompatibility of 1-hydroxy-4,5-dimethyl-imidazole 3 oxide. However, the coformer failed to give a significant increase in solubility. Salas-Zuniga et al. studied the e ff ect of hydroxypropyl methylcellulose and methylcellulose on the dissolution behaviour of two nitazoxanide cocrystals [ 11 ]. Using polymer-based powder formulations of nitazoxanide-succinic acid, they achieved a significant improvement of the apparent solubility of nitazoxanide compared to formulations of the pure API. It was suggested that the solubility enhancement was due to a polymer-induced delay of nucleation and crystal growth. Witika et al. report a cocrystal of lamivudine and zidovudine [ 12 ]. The dual-drug cocrystal was characterised by X-ray powder di ff raction, Raman spectroscopy, FT-IR spectroscopy, di ff erential scanning calorimetry and energy-dispersive X-ray spectroscopy. Surfactants were applied to produce and stabilise nano-cocrystals with specific, pre-defined critical quality attributes such as particle size, polydispersity index, and zeta potential to exploit the advantages of nano-sized drug delivery systems. In a follow-up paper, the same authors describe a Design of Experiment approach to optimise the cold sonochemical synthesis of the lamivudine-zidovudine nano-cocrystals in the presence of surfactants and polymers [ 13 ]. The nano-cocrystals proved to be less cytotoxic in HeLa cells than a physical mixture of the two APIs. Funding: This research received no external funding. Conflicts of Interest: The author declares no conflict of interest. References 1. Karagianni, A.; Malamatari, M.; Kachrimanis, K. Pharmaceutical Cocrystals: New Solid Phase Modification Approaches for the Formulation of APIs. Pharmaceutics 2018 , 10 , 18. [CrossRef] [PubMed] 2. Karimi-Jafari, M.; Padrela, L.; Walker, G.M.; Croker, D.M. Creating Cocrystals: A Review of Pharmaceutical Cocrystal Preparation Routes and Applications. Cryst. Growth Des. 2018 , 18 , 6370–6387. [CrossRef] 3. Food and Drug Administration. Regulatory Classification of Pharmaceutical Co-Crystals, Guidance for Industry. February 2018. Available online: https: // www.fda.gov / media / 81824 / download (accessed on 20 August 2018). 4. Williams, H.D.; Trevaskis, N.L.; Charman, S.A.; Shanker, R.M.; Charman, W.N.; Pouton, C.W.; Porter, C.J.H. Strategies to address low drug solubility in discovery and development. Pharmacol. Rev. 2013 , 65 , 315–499. [CrossRef] [PubMed] 5. Springuel, G.; Leyssens, T. Innovative Chiral Resolution Using Enantiospecific Co-Crystallization in Solution. Cryst. Growth Des. 2012 , 127 , 3374–3378. [CrossRef] 6. Machado Cruz, R.; Boleslavsk á , T.; Ber á nek, J.; Tieger, E.; Twamley, B.; Santos-Martinez, M.J.; Dammer, O.; Tajber, L. Identification and Pharmaceutical Characterization of a New Itraconazole Terephthalic Acid Cocrystal. Pharmaceutics 2020 , 12 , 741. [CrossRef] 7. Nugrahani, I.; Kumalasari, R.A.; Auli, W.N.; Horikawa, A.; Uekusa, H. Salt Cocrystal of Diclofenac Sodium-L-Proline: Structural, Pseudopolymorphism, and Pharmaceutics Performance Study. Pharmaceutics 2020 , 12 , 690. [CrossRef] [PubMed] 2 Pharmaceutics 2020 , 12 , 834 8. Buol, X.; Robeyns, K.; Caro Garrido, C.; Tumanov, N.; Collard, L.; Wouters, J.; Leyssens, T. Improving Nefiracetam Dissolution and Solubility Behavior Using a Cocrystallization Approach. Pharmaceutics 2020 , 12 , 653. [CrossRef] [PubMed] 9. Kale, D.P.; Puri, V.; Kumar, A.; Kumar, N.; Bansal, A.K. The Role of Cocrystallization-Mediated Altered Crystallographic Properties on the Tabletability of Rivaroxaban and Malonic Acid. Pharmaceutics 2020 , 12 , 546. [CrossRef] [PubMed] 10. Wr ó blewska, A.; ́ Sniechowska, J.; Ka ́ zmierski, S.; Wielgus, E.; Bujacz, G.D.; Mlosto ́ n, G.; Chworos, A.; Suwara, J.; Potrzebowski, M.J. Application of 1-Hydroxy-4,5-Dimethyl-Imidazole 3-Oxide as Coformer in Formation of Pharmaceutical Cocrystals. Pharmaceutics 2020 , 12 , 359. [CrossRef] [PubMed] 11. Salas-Z ú ñiga, R.; Rodr í guez-Ruiz, C.; Höpfl, H.; Morales-Rojas, H.; S á nchez-Guadarrama, O.; Rodr í guez-Cuamatzi, P.; Herrera-Ruiz, D. Dissolution Advantage of Nitazoxanide Cocrystals in the Presence of Cellulosic Polymers. Pharmaceutics 2020 , 12 , 23. [CrossRef] [PubMed] 12. Witika, B.A.; Smith, V.J.; Walker, R.B. A Comparative Study of the E ff ect of Di ff erent Stabilizers on the Critical Quality Attributes of Self-Assembling Nano Co-Crystals. Pharmaceutics 2020 , 12 , 182. [CrossRef] [PubMed] 13. Witika, B.A.; Smith, V.J.; Walker, R.B. Quality by Design Optimization of Cold Sonochemical Synthesis of Zidovudine-Lamivudine Nanosuspensions. Pharmaceutics 2020 , 12 , 367. [CrossRef] [PubMed] © 2020 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http: // creativecommons.org / licenses / by / 4.0 / ). 3 pharmaceutics Article Identification and Pharmaceutical Characterization of a New Itraconazole Terephthalic Acid Cocrystal Ricardo Machado Cruz 1 , Tereza Boleslavsk á 2,3 , Josef Ber á nek 2 , Eszter Tieger 2 , Brendan Twamley 4 , Maria Jose Santos-Martinez 1,5 , Ondˇ rej Dammer 2 and Lidia Tajber 1, * 1 School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Dublin 2, Ireland; cruzr@tcd.ie (R.M.C.); santosmm@tcd.ie (M.J.S.-M.) 2 Zentiva, k.s., U Kabelovny 130, 102 37 Prague, Czech Republic; tereza.boleslavska@zentiva.com (T.B.); josef.beranek@zentiva.com (J.B.); tiegereszter@gmail.com (E.T.); ondrej.dammer@zentiva.com (O.D.) 3 Department of Chemical Engineering, University of Chemistry and Technology, Prague, Technick á 5, 166 28 Prague, Czech Republic 4 School of Medicine, Trinity College Dublin, Dublin 2, Ireland; twamleyb@tcd.ie 5 School of Chemistry, Trinity College Dublin, Dublin 2, Ireland * Correspondence: ltajber@tcd.ie; Tel.: + 353-1-896-2787 Received: 17 July 2020; Accepted: 31 July 2020; Published: 6 August 2020 Abstract: The crystallization of poorly soluble drug molecules with an excipient into new solid phases called cocrystals has gained a considerable popularity in the pharmaceutical field. In this work, the cocrystal approach was explored for a very poorly water soluble antifungal active, itraconazole (ITR), which was, for the first time, successfully converted into this multicomponent solid using an aromatic coformer, terephthalic acid (TER). The new cocrystal was characterized in terms of its solid-state and structural properties, and a panel of pharmaceutical tests including wettability and dissolution were performed. Evidence of the cocrystal formation was obtained from liquid-assisted grinding, but not neat grinding. An e ffi cient method of the ITR–TER cocrystal formation was ball milling. The stoichiometry of the ITR–TER phase was 2:1 and the structure was stabilized by H-bonds. When comparing ITR–TER with other cocrystals, the intrinsic dissolution rates and powder dissolution profiles correlated with the aqueous solubility of the coformers. The rank order of the dissolution rates of the active pharmaceutical ingredient (API) from the cocrystals was ITR–oxalic acid > ITR–succinic acid > ITR–TER. Additionally, the ITR–TER cocrystal was stable in aqueous conditions and did not transform to the parent drug. In summary, this work presents another cocrystal of ITR that might be of use in pharmaceutical formulations. Keywords: itraconazole; terephthalic acid; cocrystal; crystal structure; mechanochemistry; solid-state; thermal analysis; wettability; dissolution 1. Introduction A pharmaceutical cocrystal can be defined as a multicomponent crystal wherein at least one component is the active pharmaceutical ingredient (API) and is in a well-defined stoichiometric ratio and bonded by non-covalent interactions with the other component(s), i.e., the coformer(s) [ 1 , 2 ]. These interactions are mainly intermolecular hydrogen bonds between functional groups forming supramolecular synthons, and moreover, additional weaker interactions such as van der Waals forces, π -stacking or halogen bonds may also help to stabilize the cocrystal structure [ 3 , 4 ]. The purpose of synthesizing pharmaceutical cocrystals is to improve the characteristics of APIs, such as the aqueous solubility, dissolution rates and stability [5]. Itraconazole (ITR) is an API with a broad-spectrum antifungal activity used for the treatment of topical and systemic mycoses as well as for prophylaxis in immunosuppressed patients [ 6 , 7 ]. ITR, due to a low aqueous solubility but a high systemic absorption, is considered a class II drug according Pharmaceutics 2020 , 12 , 741; doi:10.3390 / pharmaceutics12080741 www.mdpi.com / journal / pharmaceutics 5 Pharmaceutics 2020 , 12 , 741 to the biopharmaceutics classification system (BCS) [ 8 ]. Indeed, ITR has a very low solubility in water (around 1 ng / mL), which increases in an acidic medium (4 μ g / mL) [ 9 ], thus the poor solubility is the limiting factor for ITR absorption. To improve the poor physicochemical properties, ITR can be converted into disordered forms such as liquid crystalline [ 10 – 12 ] and amorphous structures, as well as polymer-based solid dispersions [ 12 ]. The commercial solid dosage formulation of ITR, Sporanox ® , is available as pellets enclosed in oral capsules [ 13 ]. The pellets are manufactured by spray-layering a solution of ITR and hydroxypropyl methylcellulose (HPMC) on sucrose beads. Then, the drug-coated beads are treated with a seal coating polymer layer (polyethylene glycol (PEG) 20,000 Da) to prevent the sticking of the beads. The API in these pellets is in the amorphous state [ 13 ]. Since the physical stability of drug in an amorphous phase can be short-lived, a crystalline, but a better soluble form of ITR would be preferred. The development of ITR cocrystals was first achieved combining this API with aliphatic dicarboxylic acids used as coformers, including fumaric acid, succinic acid, l -malic acid, l -tartaric acid, d -tartaric acid and d , l -tartaric acid [ 14 ]. Remenar’s work reported a remarkable improvement of the ITR dissolution with an approximately 20-fold enhancement of the cocrystals prepared with l -malic and l -tartaric acids, which had dissolution profiles comparable to the commercial form of ITR, Sporanox ® capsules. In another work, Shevchenko et al. [ 15 ] produced ITR cocrystals with coformers of di ff erent carbon chain lengths and determined that linear aliphatic dicarboxylic acids should have no more than seven carbon atoms in their chains to successfully form a cocrystal with ITR. That work also demonstrated that cocrystallisation is an e ffi cient approach to improve the dissolution rates of ITR. However, a recent study presented that an ITR cocrystal with an aliphatic dicarboxylic acid comprising more than seven carbon atoms, suberic acid, can be prepared by rapid solvent evaporation and spray drying, resulting in the material being able to dissolve rapidly. This cocrystal had a 39 times faster intrinsic dissolution rate than the crystalline ITR [16]. Little is published on the structural analysis of ITR cocrystals, nevertheless, such studies on the ITR and succinic acid (SUC) cocrystal revealed that this form has a trimeric building block, where two molecules of the API are oriented antiparallel to one another and bridged by the coformer. The main interactions found to be responsible for the intermolecular arrangement of this structure were H-bonds formed by the hydroxyl moiety located on both sides of the acid and one of the nitrogen atoms of the 1,2,4-triazole ring of each ITR molecule (Figure 1) [14]. ( a ) ( b ) Figure 1. Molecular structures of ( a ) itraconazole (ITR) and ( b ) terephthalic acid (TER). Terephthalic acid (TER) is a benzenedicarboxylic acid with the carboxylic groups attached in positions one and four (Figure 1). TER has very low toxicity and has been employed in the cocrystallization of a few APIs, such as apovincamine [ 17 ], betulin [ 18 ], isoniazid [ 19 ] and gabapentin [ 20 ]. Considering that most investigations on the identification of ITR cocrystals are limited 6 Pharmaceutics 2020 , 12 , 741 to aliphatic dicarboxylic acids [ 14 – 16 , 21 ], coformers with other architecture appears to be unexplored. Therefore, this work carried out an experimental screening aiming to identify a possible new cocrystal of ITR combining this API with TER to ascertain the relevant molecular elements in the coformers that enable the formation of new cocrystal phases of ITR. This new cocrystalline form was extensively evaluated regarding its solid-state characteristics, as was the impact ITR cocrystallisation had on a range of pharmaceutical properties. Finally, the cocrystal dissolution was studied including an intrinsic dissolution study, a simple mixture with lactose as well as a mixture comprising excipients used in the commercial ITR formulation, comparing the performance of the new cocrystal with those based on aliphatic dicarboxylic acids. 2. Materials and Methods 2.1. Materials Itraconazole (ITR) was purchased from Glentham Life Sciences Ltd. (Whitshire, UK). MeOH (HPLC grade) and terephthalic acid (TER) were purchased from Sigma-Aldrich (Arklow, Ireland). All other ingredients, such as solvents, solution and bu ff er components, as well as polymers, were kindly provided by Zentiva (Prague, Czech Republic). 2.2. Methods 2.2.1. Neat Grinding (NG) and Liquid-Assisted Grinding (LG) For these preparations, 40 mg of ITR and an amount of TER corresponding to 2:1, 1:1 and 1:2 of API-coformer molar ratios were carefully weighted. Afterwards, the compounds were ground for 30 s using an agate mortar and pestle. For liquid-assisted grinding, two drops of methanol were added to the mixture of powders. 2.2.2. Cocrystallisation by Slurrying In this method, solutions of ITR at 0.74 and 2.20 mg / mL were prepared in methanol (MeOH) and acetone, respectively. Then, a 1 mL aliquot of each solution was transferred to a 1.5 mL glass vial and a quantity of TER was added in a 1:50 API-coformer molar ratio. Then, the hermetically closed vials containing the suspensions were mixed at 50 ◦ C for 8 h and thereafter for next 4 days at room temperature using an IKA RT 15 magnetic stirrer (Germany). Afterwards, the slurries were dried at 30 ◦ C and 100 mbar in a 3608-6CE vacuum oven (ThermoFisher Scientific ™ , Waltham, MA, USA). 2.2.3. Cocrystallisation by Ball Milling (BM) A quantity of 300 mg of ITR was weighted and added to an amount of each coformer (oxalic acid, succinic acid and TER) corresponding to a 2:1 API-coformer molar ratio. Then, the powders were transferred to a 25 mL stainless-steel grind jar containing two 15 mm stainless-steel balls. Before grinding, two drops of acetone were added to the powders. The mixtures were ground in two cycles of 10 min at 25 Hz using a Retsch Mixer Mill MM 200 (Haan, Germany). 2.2.4. Cocrystallisation by Slow Evaporation A solution of ITR and TER was prepared by weighing 5.96 mg of the API and 0.70 mg of the coformer to prepare a 2:1 mole / mole mixture of the components. The powders were transferred into a glass vial and solubilized in 10 mL of methanol by sonication in a U300 H ultrasonic bath (Ultrawave, Rumney, UK) to obtain a clear solution. Then, the solution was filtered using a 0.45 μ m PTFE syringe filter (Fisher Scientific, Loughborough, UK). Parafilm was used to cover the top of the vial and small holes were pierced to allow for solvent evaporation. The solution was left at room conditions until crystallization occurred. 7 Pharmaceutics 2020 , 12 , 741 2.2.5. Freeze Drying of Itraconazole (ITR) Firstly, a 10 mg / mL solution of ITR in dioxane was prepared by weighing 1 g of the API and solubilizing it in 100 mL of the solvent. The solution was divided into three round-bottomed flasks and frozen using liquid nitrogen while rotating using a Rotavapor R-205 (Büchi, Flawil, Switzerland). The samples were dried for 18 h using a freeze drier, ALPHA 2-4 LSC (Martin Christ, Osterode am Harz, Germany), with manifolds for a connection of NS 29 / 32 flasks under a vacuum of 2 × 10 − 3 mbar , between 5 and 6 m 3 / h suction and ice condenser adjusted to − 85 ◦ C. No secondary drying step was applied. 2.2.6. Di ff erential Scanning Calorimetry (DSC) A thermal analysis was performed by carefully weighting dried samples and placing them in 40 μ L aluminum pans that were sealed with a lid containing three vent holes. The samples were subjected to DSC runs in a temperature ranging from 25 to 400 ◦ C with a heating rate of 10 ◦ C / min using a Mettler Toledo DSC 822 e / 700 (Greifensee, Switzerland) under nitrogen purge [ 22 ]. An empty aluminum pan was used as a reference. The equipment was calibrated with an indium standard. 2.2.7. Powder X-ray Di ff raction (PXRD) PXRD patterns were obtained with a laboratory X’PERT PRO MPD (PANalytical, Almelo, Netherlands) di ff ractometer with CuK α ( λ = 1.542 Å) radiation. The generator was operated at an excitation voltage of 45 kV and anodic current of 40 mA. The following scan parameters were utilized: scan type—gonio, measurement range of 2–40 ◦ 2 θ , step size of 0.02 ◦ 2 θ and the time per step was 200 s. The samples were placed on a zero-background silica sample holder. Alternatively, PXRD measurements were performed using a Rigaku Miniflex II, desktop X-ray di ff ractometer (Tokyo, Japan) equipped with a CuK α ( λ = 1.54 Å) radiation X-ray source. Dried samples were mounted on a low-background silicon sample holder and scanned over a 2 θ range of 2–40 degrees [23]. 2.2.8. Single Crystal X-ray Analysis A monocrystal of ITR–TER with approximate dimensions of 0.030 mm × 0.140 mm × 0.150 mm was used for the X-ray crystallographic analysis. The X-ray intensity data were measured at 100 ± 2 K on a Bruker Apex Kappa Duo (Billerica, MA, USA) with an Oxford Cobra Cryosystem low-temperature device (Oxford, UK) using a MiTeGen micromount (Ithaca, NY, USA). Bruker APEX software was used to correct for Lorentz and polarization e ff ects. The crystallographic data were analyzed using Mercury 2020.1 and CrystalExplorer (ver. 17.5) [24] software. 2.2.9. Fourier-Transform Infrared Spectroscopy (FTIR) and Raman Spectroscopy The dried powders were subjected to FT–IR spectroscopy on a PerkinElmer Spectrum 100 (Waltham, MA, USA), equipped with a universal attenuated total reflection (ATR) device and a ZnSe crystal. The FT–IR spectra of the samples were recorded in a wavelength range from 500 to 4000 cm − 1 The spectra were acquired by averaging 10 scans taken with a resolution of 4 cm − 1 The Raman spectra of powders were measured directly in glass vials using a Raman Spectrometer RFS 100 / S (Bruker, Billerica, MA, USA). The spectra were acquired by averaging 64 scans taken with a resolution of 4 cm − 1 and laser power of 250 mW. 2.2.10. Morphological Analysis A Zeiss Supra variable Pressure Field Emission Scanning Electron Microscope (SEM, Ulm, Germany) equipped with a secondary electron detector and an accelerating voltage of 5 kV was used for the morphological examination. The produced powder samples were placed on carbon tabs attached to aluminum stubs and sputter coated with gold / palladium under vacuum before analysis [10]. 8 Pharmaceutics 2020 , 12 , 741 2.2.11. Contact Angle Measurements Powdered samples were compacted into 4.5 cm in diameter disks and placed on the lifting table of a Drop Shape Analyzer DSA 25 (Krüss, Hamburg, Germany). Then, an automated dosing syringe containing water at 20 ◦ C was used to deposit a single drop of 14 μ L on the surface of the disks. The images of the water drop on the surface of the disks were recorded for 10 min by a high-resolution camera and processed by ADVANCE software ver. 1.9 (Krüss) to calculate the contact angle. Each sample had the contact angle measured in duplicate. 2.2.12. Intrinsic Dissolution Rate (IDR) Study Disks 8 mm in a diameter were prepared by compressing 50 ± 2 mg of powder in a stainless-steel cylindrical die system at approximately 100 kg / cm 2 for 120 s. Then, the opposite side of the steel die was sealed using a rubber plug, leaving a 0.503 cm 2 surface of the disk exposed. The stainless-steel dies were used as the disk holders and were loaded automatically by the robotic arm of the Pion inForm (Pion, Forest Row, UK) and immersed in the dissolution media. For each test, 40 mL of the medium (acetate-phosphate bu ff er comprising 150 mM NaCl) was preheated to 37 ◦ C and the pH was adjusted with 0.5 M HCl to 1.2. The agitation was set to 100 rpm. A spectral scan (190–720 nm) was collected every 30 s and the concentration was calculated against the calibration curve obtained previously under identical conditions. 2.2.13. Dissolution Analysis 2.2.13.1. Powder Dissolution of ITR Systems Mixed with Lactose For this procedure, samples were prepared by weighing an amount of ITR (starting material), freeze dried ITR (FD ITR), ITR–TER, itraconazole-oxalic acid cocrystals (ITR–OXA) and itraconazole-succinic acid cocrystals (ITR–SUC) containing an equivalent of 100 mg of ITR and mixing with lactose monohydrate in a 1:6 w / w API:excipient ratio to allow wettability and dispersibility in the liquid medium. Sporanox ® was used as the reference formulation and the pellets were removed from the capsule before the use in the dissolution experiments. The dissolution analysis was performed using a solution prepared by mixing 33 mM NaCl with 67 mM HCl (artificial gastric juice (AGJ)) containing 0.05% ( v / v ) of Tween 20. The pH of this mixture was then adjusted to 1.2 with 0.5 M HCl. The experiments were carried out using a standard USP II dissolution apparatus (Sotax, Aesch, Switzerland) attached to a UV-Vis spectrophotometer Specord 200 Plus (Analytik Jena, Jena, Germany). The powders were added directly to the media (900 mL) kept at 37 ◦ C and agitated at 75 rpm for the first 45 min and at 150 rpm for the final 15 min. The aliquots were automatically taken at predefined time points (2, 5, 10, 15, 20, 25, 30, 45, 50 and 60 min) and the ITR concentration was assessed by measuring the absorbance at 255 nm. 2.2.13.2. Powder Dissolution of ITR Systems Mixed with Other Excipients A second dissolution test evaluated the dissolution of the API when physically mixed with the same excipients as those present in Sporanox ® (Jassen, Beerse, Belgium). For this purpose, 350 mg of ITR (starting material) and FD ITR or the amount of the ITR–TER, ITR–OXA and ITR–SUC cocrystals containing the equivalent of 350 mg of the API were carefully weighted and mixed with the other excipients in the concentrations listed in Table 1 for 5 min in 200 mL plastic bottles using a Turbula ® mixer (WAB group, Muttenz, Switzerland). The dissolution analysis was carried out as described in Section 2.2.13.1, using 900 mL peak vessels in a dissolution apparatus 708-DS (Agilent, Lexington, MA, USA) coupled to an Agilent UV-Vis spectrophotometer Cary 60. 9 Pharmaceutics 2020 , 12 , 741 Table 1. Composition of the powders used in the dissolution study. Excipient (%, w / w ) Formulation ITR FD ITR ITR–OXA ITR–SUC ITR–TER API 21.74 21.74 Cocrystal 23.22 23.13 23.70 Sucrose 41.74 41.74 40.95 41.0 40.69 HPMC ( * ) 32.61 32.61 31.99 32.03 31.79 PEG ( ** ) 3.91 3.91 3.84 3.84 3.82 Total (%) 100 100 100 100 100 Powder weight (mg) ( *** ) 460 460 501.1 498.32 514.2 (*) Hydroxypropyl methyl cellulose 2910 (5 mPa.s (HPMC)); (**) polyethylene glycol 6000 Da (PEG); (***) mass of the powder containing 100 mg of ITR used in the dissolution test. 2.2.14. Statistical Analysis The statistical analysis of the data was carried out using GraphPad Prism ® for Windows, version 5.01, applying a Student’s t -test or one-way ANOVA with Tukey’s post-test with 95% of confidence when appropriate. Statistical significance was when p < 0.05. 3. Results and Discussion 3.1. Characterization of ITR and Terephthalic Acid (TER) Mixtures Following Neat and Liquid-Assisted Grinding Mechanochemical methods of cocrystal production have gained a considerable interest in recent times [ 25 , 26 ]. Therefore, as the first approach, a neat, solvent-free (NG) grinding of ITR and TER in a few stochiometric ratios, followed by the liquid-assisted grinding (LG) was performed. The ITR “as received” material was identified as form I of itraconazole [ 27 ]. The PXRD analysis of the samples prepared by neat grinding (NG (Figure 2)) revealed that the mixtures post-processing had similar di ff ractograms to those of ITR with two Bragg peaks at 17.5 and 17.95 ◦ 2 θ , corresponding to TER. The samples prepared by LG had additional, but weak, Bragg peaks at 3.5, 7.0 and 21.2 ◦ 2 θ , which were absent in the parent materials, indicating the formation of a new phase. Interestingly, the TER component in all samples post NG and LG appeared to be at least partially amorphous, as evidenced by the weak or absent di ff raction peaks of TER. Therefore, the LG method is more e ffi cient in producing the ITR–TER cocrystal than NG most likely because the cocrystallisation process by LG was facilitated by the presence of methanol. Indeed, the use of solvents in cocrystallisation screening is common and the impact of the solvent is described as catalytic, since it is used in a very small quantity and is also not part of the final cocrystal [28]. A DSC analysis of ITR and TER on their own showed that the drug melted at around 166 ◦ C [ 12 ], while TER melted with sublimation at around 350 ◦ C [ 29 ] (Figure 3). A thermal analysis of the binary ITR–TER mixtures post-processing showed thermograms with a sharp endothermic peak at 198 ◦ C (Figure 3), which was absent in the parent compounds and indicated the melting of the new phase. However, all these mixtures also had an endothermic peak with an onset at 164 ◦ C (Figure 3), assigned to the melting of ITR. This peak in the ITR–TER 1:1 (LG) and ITR–TER 1:2 (LG) systems was almost imperceptible, with enthalpies of 2.6 and 0.7 J / g, respectively. In contrast, the ITR peak in the samples ITR–TER 1-1 (NG) and ITR–TER 1-2 (NG) had an enthalpy of 13.5 and 18.4 J / g, respectively, indicating that the methanol used in LG contributed to a greater conversion of ITR into the new phase. A small exothermic peak was detected for all NG samples and ITR–TER 1:2 (LG) immediately followed the ITR peak, consistent with the previous studies on the thermal behavior of binary mixtures where the formation of a cocrystal was observed upon heating [ 30 ]. Thus, in relation to NG mixtures, 10 Pharmaceutics 2020 , 12 , 741 the cocrystal melting peak, visible in DSC traces, is of the new form which appears upon heating. Additionally, what can be concluded from the DSC study is that a broad endothermic peak with an onset at around 310 ◦ C for the ITR–TER 1:1 samples and an onset at around 320 ◦ C for the ITR–TER 1:2 systems was detected, assigned to the excess of TER. It may suggest that the potential stoichiometry of the ITR–TER cocrystal might be 2:1. ,QWHQVLW\ DX θ GHJUHH ,75DU 7(5DU ,757(5 1* ,757(5 1* ,757(5 1* ,757(5 /* ,757(5 /* ,757(5 /* Figure 2. Powder X-ray di ff raction (PXRD) of the ITR and TER starting material as the received powders (“ar”) and binary ITR and TER mixtures following neat grinding (NG) and liquid-assisted grinding (LG). * Indicates the distinct peak of the new phase (cocrystal), broken black lines show the position of the ITR di ff raction peaks, while the broken red lines show the position of the TER di ff raction peaks. 7(5DU ,75DU +HDWIORZ -J 7HPSHUDWXUH R & ,757(5 1* ,757(5 1* ,757(5 1* ,757(5 /* ,757(5 /* ,757(5 /* H[RA Figure 3. Di ff erential scanning calorimetry (DSC) of the ITR and TER starting material, as received powders (“ar”) and binary ITR and TER mixtures following neat grinding (NG) and liquid-assisted grinding (LG). The area highlighted in grey indicates the ITR melting region, in purple the cocrystal melting range and in light red the TER melting and degradation. Arrows indicate the presence of an exothermic peak immediately following the ITR melting event. 3.2. Properties of ITR and TER Samples Made by Slurring, Evaporation and Ball Milling Methods A PXRD analysis of samples obtained by the slurring of ITR and TER in methanol and acetone was very similar to that of TER and indicated an incomplete conversion to the cocrystal, with only weak intensity Bragg peaks of the new phase visible at approximately 7.0, 10.4, 12.4 ◦ 2 θ (Figure 4). A more successful method was the slow evaporation of ITR and TER from methanol, and the di ff raction pattern of the 2:1 ITR–TER system is presented in Figure 4. A number of di ff raction peaks characteristic of the cocrystal phase can be discerned, however the peak at 15.0 ◦ 2 θ can be described as the ITR starting material powder. The method that gave the purest cocrystal, based on the PXRD results, was ball milling (Figure 4). The ITR–TER 2:1 system had a di ff raction pattern distinct from those of the starting material powders, particularly due to the peaks at 3.5, 7.0, 10.5, 12.4, 17.8, 19.3 and 21.2 ◦ 2 θ . Therefore, 11